Protein kinases are an important class of enzymes, which are frequently used as targets in drug development. Protein kinases mediate intracellular signal transduction. They do this by effecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. There are a number of kinases and pathways through which extracellular and other stimuli cause a variety of cellular responses to occur inside the cell. Examples of such stimuli include environmental and chemical stress signals (e.g. osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, reactive oxygen species like H.sub.2O.sub.2), cytokines (e.g. interleukin-1 (IL-1) and tumor necrosis factor.alpha. (TNFα)), and growth factors (e.g. insulin, insulin-like growth factor (IGF1), granulocyte macrophage colony-stimulating factor (GM-CSF), and fibroblast growth factor (FGF). An extracellular stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose and lipid metabolism, control of protein synthesis and regulation of cell cycle.
Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events. These diseases include autoimmune diseases, inflammatory diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, Alzheimer's disease or hormone-related diseases, such as diabetes.
Accordingly, there has been a substantial effort in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents. Thus, protein kinases have emerged as one third of all new targets in pharmaceutical industry. The protein kinase ATP binding site is clearly a drugable site and almost all protein kinase inhibitors target this site. Importantly, a similar effort has not been evident for the development of protein kinase non-ATP competitive inhibitors. In addition, the existence of small molecule compounds that target regulatory sites on protein kinase catalytic domains by an allosteric mechanism were for the first time described in Engel et al., EMBO J. 2006, Vol. 25, pp. 5469-5480 and EP-A-1486488. This was surprising as small molecule compounds were generally considered as being not suitable as they cannot promote the required conformational changes. Alternatively, the assays and analysis tools usually performed for screening and analysis of the data may not select for these compounds. Alternatively, compounds might have been disregarded as potential drug molecules, or the regulatory sites disregarded as possible drugable sites. At any rate, the finding and demonstration that small molecule compounds can regulate protein kinase activities by interacting with regulatory sites is not obvious.
Although the ATP binding site is a proven drugable site, small molecule compounds which are directed against the ATP binding site of a given protein kinase, have a high probability to bind to ATP binding site(s) of one or more other protein kinases, or even other ATP binding enzymes such as DNA polymerases or pyruvate kinase. The reason for this is that binding sites for the universal enzymatic co-factor ATP share strong homologies, similar dimensions and shapes, which enable binding of ATP but potentially also ATP-competitive inhibitors, to an undefined number of ATP-binding enzymes. Such non-target related inhibition will inevitably lead to side effects if not amended. The improvement of selectivity while maintaining potency by means of medicinal chemistry can be a difficult, time-consuming process, which in many cases fails in the end.
In addition, ATP-competitive compounds are often found to be considerably less potent in cells and organs due to the high intracellular ATP levels, which are in the range of 1-2 mM, when compared to cell-free assay conditions which usually work with ATP concentrations of 10-20 μM or less. Thus, a drop of IC50 values of up to 100 fold must be expected when compounds are transferred to cell assays.
Up to now, the problem described here was addressed by the extension of merely ATP-competitive compounds, i.e. additional chemical moieties were added to the ATP-competitive scaffolds which were designed to interact with additional, less strongly conserved amino acid residues located at the close environment of the ATP-binding site. Such compounds possess a non-ATP competitive portion but are not truly “allosteric”. By doing so, it was possible in some cases to specifically stabilize a catalytically less competent form of the kinase. In this low number of cases, it was possible to develop rather selective compounds. A well known example is imatinib (gleevec) from Novartis, which inhibits, besides the actual target BCR-Abl tyrosin kinase, only a limited number of other tyrosine kinases, such as c-kit and platelet-derived growth factor receptor (PDGFR) (Adrián, F. J. et al., Nature Chemical Biology 2:95-102 (2006)).
Another example for an inhibitor which enganges a binding site adjacent to the ATP binding site, thereby stabilizing an inactive conformation, is the p38 MAP kinase inhibitor BIRB 796 (Pargellis, C. et al., Nature Structural Biology 9:268-272 (2002)). Since the size of orally available compounds is limited to below 500 Da, this strategy can only be successful if differences outside but within reach of the ATP binding site, in particular between close homologs of protein kinases, are big enough to allow for creation of selectivity. This limitation will prevent larger applications of this strategy in the kinase drug discovery field, though it might work for a subset of current kinase targets. However, it is unlikely to work for targeting such closely related isoforms such as the PKC or PKB family of kinases.
However, true allosteric binding at sites remote from the ATP site that affect kinase activity have barely been described (for example, Akt-I-1, a pleckstrin-homology-domain-dependent Akt (PKB) inhibitor (Barnett, S. F. et al., Biochem. J. 38:399-408 (2005)). Review articles summarising the efforts to develop kinase inhibitors termed “allosteric” actually refer to ATP-competitive compounds with a non-ATP-competitive portion of interaction in the majority of cases (see Kiselyov et al., Recent Progress in Development of Non-ATP Competitive Small-Molecule Inhibitors of Protein Kinases, Mini-Reviews in Medicinal Chemistry, 2006, 6, 109-120). Furthermore, Gumireddy et al. (P.N.A.S. USA, 2005, vol. 102, pp. 1992-1997) report a non ATP-competitive BCR-Abl inhibitor which however does not act allosterically but in a substrate competitive manner. The same group also disclosed ON01910, a non ATP-competitive inhibitor of Polo-like kinase-1, which also displayed substrate competitive binding (Gumireddy et al., Cancer Cell. 2005, 7, 275-286). Non ATP-competitive inhibitors have also been described for PKB (AKT), which bind to the kinase in a PH domain-dependent fashion, indicating that they are not only targeting the catalytic domain (reviewed in Martinez et al, Current Topics in Medicinal Chemistry 2005, 5, 109-125). Further non ATP-competitive PKB/AKT inhibitors with unknown binding sites were identified in screening campaigns (reviewed in Martinez et al, Current Topics in Medicinal Chemistry 2005, 5, 109-125, and Amaravadi and Thompson, J. Clin. Invest. 2005, 115, 2618-2624). Furthermore, thiadiazolidinones have been disclosed as non ATP-competitive inhibitors of GSK-3beta; the binding site has not been determined experimentally (Martinez et al., J. Med. Chem. 2002, 45, 1292-1299, and WO/2005/097117)).
An obvious limitation of the vast majority of compounds whose binding sites are at least overlapping with the ATP binding site is that they can usually only inhibit the enzymatic activity but not activate. In the current state of the art, activation of protein kinase regulated signalling pathways is only possible indirectly, e.g. by the following ways:                receptor agonists: in cases where the protein kinase is activated following to receptor stimulation, agonistic compounds can be developed. This route is often employed in drug development but is of course dependent on the expression of the appropriate receptor proteins in the target organs, and is not applicable to cases where the stimulus is not properly propagated from the receptor to the executive protein kinase. The latter situation is encountered e.g. with diabetes type 2, where resistance to insulin is mainly mediated trough inhibitory phosphorylation of the IRS proteins (insulin receptor substrate), which is impairing signal transduction from the insulin receptor.        activation of protein kinases possible in some cases by abolishing the inhibition imposed by regulatory subunits. A known example is cAMP-dependent protein kinase (PKA), which is released in an active state from a complex with regulatory subunits after binding of cAMP to said regulatory proteins. In this case, cAMP mimetics could tigger the same effect. The applicability of such strategies depends on particular regulatory mechanisms reserved to only a few kinases and might provoque another type of selectivity problems since second messengers usually have pleiotropic effects due to affecting several target proteins.        inhibition of negative feedback pathways: a known example herefor is the PDK1/PKB pathway in insulin responsive cells. Via inhibition of ribosomal S6-Kinase the negative feedback inhibition of insulin signalling is abolished, thus effecting enhanced activity of PDK1 and stronger phosphorylation and activity of e.g. PKB, which is a major mediator of insulin signalling. As a consequence, the insulin action is enhanced and prolonged. Therefore, rapamycin and analogous, which cause inhibition of S6Kinase, are also tested for the indication diabetes type 2 as insulin sensitizers. ATP competitive inhibitors of protein kinases such as S6Kinase will show the described selectivity problems again, while in particular rapamycin and analogues, albeit non-ATP competitive, mediate further effects besides inhibition of the S6Kinase resulting in immunosuppression, which is not favourable during a lifetime treatment of a chronic disease like diabetes type 2.        
At any rate, little reports or patents disclose small molecule compounds which are able to incease the catalytic activity of a given protein kinase by binding to the catalytic domain. A rare example is the recent report on an in vitro-activator of Aurora kinase A (Kishore et al., J. Med. Chem. 2008, 51, 792-797). The lack of availability of such compounds and the lack of knowledge of drugable allosteric binding sites allowing the development of such compounds, respectively, have hitherto led to a disregarding of potential applications for such compounds. It is therefore expected that on the basis of our allosterically activating compounds, further indications with a medical need to activate a target kinase will be identified and met by utilising compounds of the present invention. Therefore, the examples 2 and 3 (Tables 2 and 3) in connection with example 6 as described below for the PDK1/PKB pathway are by far not limiting but rather reflect current state of the art which is dominated by concepts for direct inhibition of protein kinase activity rather than activation.
AGC kinases form a group within the protein kinase superfamily (Manning et al., Science 2002, 298, 1912-1934). AGC kinase homologues are found throughout the whole eukaryotic world. The AGC kinase group consist of 63 protein kinase domains from which 6 are predicted to be pseudogenes. AGC kinase group can be divided into several families according to their homology within the catalytic domain. Furthermore, they can be grouped and named according to the most relevant protein kinases members of each group. The AGC kinases can be divided into families according to the Protein kinome, the families are: AKT (PKB), LAT, ROCK, MRCK, DMPK, GRK, MAST, NDR, PDK, PKA, 4 PKC families, PKG, PKN, 4RSK families, RSKL, SGK, YANK. Each one of these families may contain subfamilies. When the tree of AGC kinases is observed, important branches within the AGC group are formed by PKC, PKB (AKT)/SGK, S6K/RSK/MSK, GRK, ROCK/DMPK/LATS/NDR, MAST, and RSKL families.
Another group of kinases, which can also be targeted by the compounds according to the present invention, are the Aurora family of kinases, Aurora-A, -B and -C. Although the Aurora kinases are not directly classified as AGC kinases, they are closely related in sequence homology and moreover, in mechanisms of regulation. This is already reflected by the fact that the Aurora kinases were placed on the same branch of the kinome as the AGC-kinase family. Being key regulators of mitosis, the Aurora kinases are exploited as pharmaceutical targets for the development of anti-cancer drugs (Carvajal et al., Clinical Cancer Research Vol. 12, 2006, pp. 6869-6875).
AGC kinases conservation throughout evolution is reflected by their overall catalytic domain sequence conservation and importantly also by its mode of regulation. Their active conformation is regulated by the state of phosphorylation of their activation loop and to secondary phosphorylations in segments outside the catalytic domain. AGC kinases have phosphorylations within the C-terminal extension to the catalytic core, which interact with the catalytic domain. Most notably is the phosphorylation within a hydrophobic motif C-terminal to the catalytic domain, which also participates in protein kinase activation. The lack of phosphorylation in this site helps to keep the protein kinase inactive in some cases, like S6K members. The mechanism by which the AGC kinases are activated upon hydrophobic motif phosphorylation appears to involve the interaction of the phosphate with a phosphate binding site, while the hydrophobic motif interacts with a hydrophobic PIF pocket (Yang, J. et al., Mol. Cell. 9:1227-40 (2002); Yang, J. et al., Nat. Struct. Biol. 9:940-4 (2002); Biondi, R. M. et al., Embo J. 19:979-88 (2000); Biondi, R. M. et al. Embo J. 21:4219-28 (2002); Frodin, M. et al., Embo J. 21 (2002)). The hydrophobic PIF pocket on its own can modulate protein kinase activity (Biondi, R. M. et al., Embo J. 19:979-88 (2000)). The role in protein kinase activation was first characterised on PDK1, by homology with PKA. By homology modelling it was found to be present and play a role on a number of AGC kinases (Frodin, M. et al., Embo J. 21:5396-407 (2002)). Furthermore, PDK1 and PKB crystal structures support the general existence within AGC kinases of a site homologous to the site in PKA that interacts with its Phe-X—X-PheCOOH C-terminal sequence (Yang, J. et al., Nat. Struct. Biol. 9:940-4 (2002); Biondi, R. M. et al. Embo J. 21:4219-28 (2002)).
A subgroup of the AGC family of protein kinases, here referred to as the “growth factor-activated AGC kinases” is activated by insulin, growth factors, many polypeptide hormones and other extracellular stimuli. This group regulates cellular division, growth, differentiation, survival, metabolism, motility and function and it includes the kinases: protein kinase B (PKBα-γ or AKT1-3), p70 ribosomal S6 kinase (S6K1,2), p90 ribosomal S6 kinase (RSK1-4), mitogen- and stress-activated protein kinase (MSK1,2), serum- and gluticocoid-induced kinase (SGK1-3) and several members of the protein kinase C (PKC).
The regulatory PIF-pocket site of the protein kinase PDK1 is a target site of phosphorylation-dependent conformational changes induced by some of the small compounds described in this application. It is envisaged that the compounds targeting this site on PDK1 may be employed for the treatment of cancers since they are expected to block the activation of protein kinases which are involved in cancers, such as S6K, RSK, SGK, PKCs, etc. It is expected that to achieve such results, the PIF-pocket of PDK1 may require to be blocked in a constitutive manner; for this, it is preferred that small compounds with slow off-rate are selected and developed into drugs. However, it can be envisaged that transient blockage of the pocket, may block transient activation of the substrate S6K and is expected to block a feed-back loop phosphorylation of IRS1; in such scenario, the block of PDK1 PIF-pocket may sensitize cells for insulin signalling. Compounds acting in this way may be selected for treatment of insulin resistance or diabetes. It is further envisaged that such compounds may be of use in other circumstances where blocking of transient PDK1 PIF-pocket-dependent phosphorylations may be required. It is expected that treatment for insulin resistance or diabetes may not require complete blockage of the pocket in a constitutive manner, but rather with a transient pharmacological profile that would favour the action of insulin after food intake.
Growth factor-activated AGC kinases as drug targets. The growth factor-activated AGC kinases are known or assumed to be important in a variety of important human diseases, and several of the kinases are reportedly included in drug development programs (e.g. PKB and PKC isoforms). Cancer: Most of the growth factor-activated AGC kinases are constitutively activated in cancer cells, due to hyperactivation of upstream activating pathways, and are known (PKB, S6K, PKC, RSK) or thought/hypothesized (SGK, MSK) to promote cancer cell growth, survival or metastasis. Drugs that inhibit these kinases may therefore be new anti-cancer drugs. Diabetes mellitus: The activation of PKB, a key mediator of insulin metabolic regulation, is reduced in type-II diabetes due to insulin resistance. Interference with S6K (by gene knockout) protects mice from dietary-induced diabetes. Activators of PKB or inhibitors of S6K may therefore be used as anti-type-II diabetes drugs. Hypertension: Hyperactivation of SGK is thought to promote hypertension. Compounds that inhibit SGK, may be used as anti-hypertensive drugs. Tuberous sclerosis complex syndrome (TSC): Inactivating mutations in the TSC genes results in hyperactivation of S6K, which is likely important in development of TSC. Inhibitors of S6K may therefore be used to treat TSC patients, for which currently no treatment exists. Other diseases in which AGC kinase inhibitors/activators may be used include chronic inflammation/arthritis, cardiac hypertrophy, neurodegenerative disorders, ischaemic conditions, and more.
AGC kinases participate in a number of further signalling pathways, many of which are involved in disease states and conditions that may be improved in patients. A number of non limiting examples of conditions related to the different subfamilies are given below. Furthermore, a large list of conditions where protein kinases are involved are being grouped and continuously updated from available sources, such as the protein kinase resource (PKR) website (http://pkr.sdsc.edu). PKC family member inhibitors as sought after for a number of conditions including the treatment of cancer; virus infections, such as treatments of cytomegalovirus infections, and HIV infections (U.S. Pat. Nos. 6,291,446 and, 6,107,327), asthma (U.S. Pat. No. 6,103,712); pain, for example pain perception and hyperalgesia (CA-A-2,336,709); skin treatments, e.g. to inhibit Langerhans cell migration induced by the presence of an allergenic agent (AU-A-200218371); renal dysfunction, such as for treatment of renal failure, intraglomerular hypertension, inhibiting glomerolosclerosis and inhibiting glomerular intestinal fibrosis (CA-A-2323172); chronic myeloid leukaemia and cute lymphoid leukaemia (CA-A-2311736), treatment of sexual dysfunctions directed to a method for inducing endothelium dependent vasodilation, smooth muscle relaxation, e.g. penile erection, clitoral engorgement and erection (U.S. Pat. No. 6,093,709), etc. Within the subfamily including PKB and SGK, inhibitors are being searched for the treatment of disease states. SGK inhibitors (U.S. Pat. No. 6,416,759) are claimed as an antiproliferative agent, and also for treatment of diseases related to a disturbance of ion channel activity, in particular, sodium and/or potassium channels, e.g. for the regulation of blood pressure (WO02/017893). PKB inhibition is seeked for a number of conditions including the treatment of proliferative diseases and where apoptosis is wanted. PKB inhibitors have also been proposed to inhibiting restenosis after angioplasty (WO03/032809). PKB inhibitors can be used to promote apoptosis of rheumatoid arthritis synovial fibroblasts for the treatment of rheumatoid arthritis (WO02/083075). PKB activators and inhibitors may be used for regulating the level of mucin production; PKB activators can be used to treat mucin overproduction in several diseases including otitis media, chronic obstructive pulmonary disease, asthma and cystic fibrosis, otitis media infections, and chronic obstructive pulmonary disease caused by nontypeable Haemophilus influenzae (US-A-2002/0151491); S6K/RSK subfamily can be targeted for diseases where subfamily members act downstream of MAPK signalling, for example in cancer and inflammation. Rapamycin inhibits S6K as a downstream target of mTOR; thus, inhibition of S6K may be wanted to obtain part of the responses obtained with rapamycin, as immunosupressant; also it may be used to treat cancer. The subfamily of G-protein coupled receptor kinases (GRKS) can be targeted to modulate the signal intensity of G-protein coupled receptors, which form the largest family within the human genome and are important targets in drug development and therapies. Disease state and conditions that can be treated with GRKs include neurological disorders, depression, inflammation, central nervous system states, osteoporosis, immunosuppressant, hypertension, infection, hypertension, retinitis pigmentosa, cancer, asthma, cystic fibrosis, arthritis, Alzheimer, Parkinson, rheumatoid arthritis, and in general conditions treated with drugs which target G-protein coupled receptors. Within the ROCK/DMPK/LATS/NDR subfamily, ROCK inhibitors are being developed as therapeutic agents for the treatment of a number of conditions, including cancer, inflammation, as immunosuppressant, a therapeutic agent of autoimmune disease, an hypertension, a therapeutic agent of angina pectoris, a suppressive agent of cerebrovascular contraction, a therapeutic agent of asthma, a prophylactic agent of peripheral circulation disorder, a prophylactic agent of immature birth, a prophylactic agent of digestive tract infection, a therapeutic agent of osteoporosis, a therapeutic agent of retinopathy and a brain function improving drug (U.S. Pat. No. 6,218,410).
PDK1 is being targeted for the treatment of cancer with an ATP competitive inhibitor termed UCN-01.
Activators of AGC kinases could also be used in therapeutics. For example, DMPK activators could be used for treatment of conditions where DMPK activity is reduced, including Myotonic Dystrophy. Furthermore, transient activation of PDK1 or PKB beta could be used to mimic insulin signalling action for the treatment of diabetes and states where GSK3 inhibition is required for treatment or cure of diseases. By inhibiting apoptosis, these activators may be used for treatment of diseases where apoptosis is to be avoided, such as in neurological disorders. Activators of RSK family members may compensate in part the effects in genetic diseases such as Coffin-Lowry syndrome. Therefore, RSK2 activators may be used for treatment of mental retardations or states where neurological performance is to be enhanced. Therefore, compounds that regulate (inhibit or activate) AGC kinases are important for drug development, since they could target conditions where the AGC kinase is required to be inhibited or activated.
Modulators of AGC kinase activities could be used as a therapy for treatment of patients with degrees of mental retardation or to enhance performances where disease states are not involved.
Phosphoinositide dependent protein kinase 1 (PDK1) and Protein kinase B (Akt/PKB) are components of an intracellular signalling pathway of fundamental importance that functions to exert the effects of growth and survival factors, and which mediates the response to insulin and inflammatory signals (Brazil, D. P. & Hemmings, B. A., Trends Biochem. Sci. 26:657-64 (2001)). PKB enzyme is rapidly activated by PDK1 phosphorylation following stimulation of phosphoinositide 3-kinase, and generation of the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate[PtdIns (3,4,5) P3].
Activated PKB phosphorylates numerous cytosolic and nuclear proteins to regulate cell metabolism, growth and survival. In the insulin signalling pathway, PKB phosphorylates GSK-3, PFK2 and mTOR, inducing glycogenesis and protein synthesis, and regulates glucose uptake by promoting the translocation of Glut4 to the plasma membrane. Cell survival and transformation are controlled by phosphorylation of BAD, caspase-9, forkhead transcription factors and IkB kinase, promoting proliferation and suppressing cell apoptosis (Datta, S. R. et al., Genes Dev. 13:2905-27 (1999)). A mechanism by which PKB stimulates cell cycle progression is by phosphorylation of the CDK inhibitors p21wAF1 and p27kiP1, causing their retention in the cytoplasm (Zhou, B. P. et al., Nat. Cell. Biol. 3:245-52 (2001)), whereas in contrast, PKB mediates nuclear localisation of mdm2 and subsequent regulation of the mdm2/p53 pathway (Mayo, L. D. & Donner, D. B., Proc. Natl. Acad. Sci. USA 98:11598-603 (2001)). In humans, the three isoforms of PKB are highly conserved, with a mean sequence identity of 73%, and share the same regulatory phosphorylation sites.
PKB plays an important role in the generation of human malignancy.
The enzyme is the cellular homologue of v-Akt, an oncogene of the transforming murine leukaemia virus PKB8 isolated from a mouse lymphoma (Staal, S. P., Proc. Natl. Acad. Sci. USA 84:5034-7 (1987)). Viral-Akt is a fusion of the viralGag protein with the PKBalpha sequence (Bellacosa, A. et al., Science 254:274-7 (1991)). Myristoylation of the Gag sequence targets v-Akt to the cell membrane, resulting in its constitutive phosphorylation. The genes for several isoforms of PKB are over-expressed and amplified in ovarian, prostate, pancreatic, gastric, and breast tumors (Testa, J. R. & Bellacosa, A., Proc. Natl. Acad. Sci. USA 98:10983-5 (2001)). Compelling evidence linking PKB to oncogenesis stems from the elucidation of the mechanism of the PTEN tumour suppressor gene. PTEN is one of the most commonly mutated genes in human cancer and somatic deletions or mutations of PTEN have been identified in glioblastomas, melanoma and prostate cancers, and are associated with increased susceptibility to breast and thyroid tumours (Cantley, L. C. & Neel, B. G., Proc. Natl. Acad. Sci. USA 96:4240-5 (1999)). PTEN negatively regulates the PI-3 kinase/PKB pathway by dephosphorylating PtdIns (3,4,5) P3 on the D-3 position, and therefore loss of PTEN activity leads to a constitutive cell survival stimulus (Maehama, T. & Dixon, J. E., J. Biol. Chem. 273:13375-8 (1998); Myers, M. P. et al., Proc. Natl. Acad. Sci. USA 95:13513-8 (1998)).
Therefore, modulators (activators or inhibitors) of PDK1 or PKB could be used for treatment of diseases, e.g. the treatment of diabetes, cancer, neurodegeneration and erectile dysfunction.
By modulating PKB activity, the phosphorylation state of Glycogen synthase kinase-3 (GSK3) could be regulated. GSK-3 is a serine/threonine protein kinase comprised of isoforms that are each encoded by distinct genes (Coghlan, M. P. et al., Chem. Biol. 7:793-803 (2000); Kim, L. & Kimmel, A. R., Curr. Opin. Genet. Dev. 10:508-14 (2000)). This enzyme participates in several signalling pathways important in disease and small molecule compounds are being developed as ATP competitive inhibitors. As these inhibitors are ATP competitive inhibitors, they inactivate GSK3 in all different pathways. As will be described below, GSK3 inhibition by compounds may also mimic Wnt signalling and promote proliferative disorders, e.g. colon cancer. As PKB does not affect the activity of GSK3 within Wnt signalling, modulation of PKB activity could be better used for a safer treatment of a number of disorders which require inhibition of GSK3, without affecting Wnt signalling. Therefore, for example, PKB .beta. activators could have the added value that they would inhibit GSK-3 downstream from PKB but not affect Wnt signalling. GSK-3 has been implicated in various diseases including diabetes, Alzheimer's disease, CNS disorders such as manic depressive disorder and neurodegenerative diseases, and cardiomyocyte hypertrophy [WO99/65897; WO00/38675; and Haq et al., J. Cell Biol. (2000) 151, 117-]. These diseases may be caused by, or result in, the abnormal operation of certain cell signaling pathways in which GSK-3 plays a role. GSK-3 has been found to phosphorylate and modulate the activity of a number of regulatory proteins. These proteins include glycogen synthase, which is the rate limiting enzyme necessary for glycogen synthesis, the microtubule associated protein Tau, the amyloid peptide, the gene transcription factor-catenin, the translation initiation factor elF2B, as well as ATP citrate lyase, axin, heat shock factor-1, c-Jun, c-Myc, c-Myb, CREB, and CEPBa. These diverse protein targets implicate GSK-3 in many aspects of cellular metabolism, proliferation, differentiation and development. In a GSK-3 mediated pathway that is relevant for the treatment of type II diabetes, insulin-induced signaling leads to cellular glucose uptake and glycogen synthesis. Along this pathway, GSK-3 is a negative regulator of the insulin-induced signal. Normally, the presence of insulin causes inhibition of GSK-3 mediated phosphorylation and deactivation of glycogen synthase. The inhibition of GSK-3 leads to increased glycogen synthesis and glucose uptake (Klein et al., PNAS, 93:8455-9 (1996); Cross et al., Biochem. J., 303:21-26 (1994); Cohen, Biochem. Soc. Trans., 21:555-567 (1993); Massillon et al., Biochem. J. 299:123-128 (1994)). However, in a diabetic patient where the insulin response is impaired, glycogen synthesis and glucose uptake fail to increase despite the presence of relatively high blood levels of insulin. This leads to abnormally high blood levels of glucose with acute and long term effects that may ultimately result in cardiovascular disease, renal failure and blindness. In such patients, the normal insulin-induced inhibition of GSK-3 fails to occur. It has also been reported that in patients with type II diabetes, GSK-3 is overexpressed [WO00/38675]. Therefore, inhibition of GSK-3 can mimic insulin action. GSK-3 activity has also been associated with Alzheimer's disease. This disease is characterized by the well-known P-amyloid peptide and the formation of intracellular neurofibrillary tangles. The neurofibrillary tangles contain hyperphosphorylated Tau protein where Tau is phosphorylated on abnormal sites. GSK-3 has been shown to phosphorylate these abnormal sites in cell and animal models. Furthermore, inhibition of GSK-3 has been shown to prevent hyperphosphorylation of Tau in cells (Lovestone et al., Current Biology 4:1077-86 (1994); Brownlees et al., Neuroreport 8:3251-55 (1997)). Therefore, it is believed that GSK-3 activity may promote generation of the neurofibrillary tangles and the progression of Alzheimer's disease. Another substrate of GSK-3 is β-catenin which is degradated after phosphorylation by GSK-3. Reduced levels of β-catenin have been reported in schizophrenic patients and have also been associated with other diseases related to an increase in neuronal cell death (Zhong et al., Nature 395:698-702 (1998); Takashima et al., PNAS 90:7789-93 (1993); Pei et al., J. Neuropathol. Exp. 56:70-78 (1997)). As a result of the biological importance of GSK-3, there is current interest in therapeutically effective GSK-3 inhibitors. Small molecules that inhibit GSK-3 have recently been reported in WO99/65897, WO02/096905 and WO00/38675.
WO 2008/019890 moreover discloses compounds having the following formula,
in whichX is selected from O, N—R and NO—R, wherein R is H, C1-4-alkyl, or -L-Y (wherein L is a linker and Y is a functional group);Q is selected from S and CH2;Z is selected from COOH, tetrazolyl, CN, phosphonic acid, phosphate and COOE (wherein E is C1-5-alkanoyloxy-C1-3-alkyl or C1-5-alkoxycarbonyloxy-C1-3-alkyl;R1, R4-R10 are selected from H, halogen, C1-4-alkyl, C2-4-alkenyl and CF3; andR2 and R3 are a member of a benzoanneleted cyclopentane, cyclohexane or benzene, or are selected from H, halogen, C1-4-alkyl, C2-4-alkenyl and CF3,as well as its use for modulating the activity of AGC kinases and for target validation studies.
Another signalling pathway in which GSK3 participates is Wnt signalling. Wnt signalling inhibits GSK3, which in turn translates into activation of transcription factors involved in tumor development, e.g. in colon cancers. Thus, GSK3 inhibition by compounds to treat diabetes or neurological disorders, in time, could lead to unwanted side effects. Importantly, PKB phosphorylation does not play a role in Wnt signalling inhibition of GSK3. Therefore, a compound triggering activation of the specific PKB isoform (PKB beta), which in turn phosphorylates and inhibits GSK3 within the insulin signalling pathway may be used to mimic insulin action for the treatment of diabetes, without affecting Wnt signalling.
One object of the invention is to provide compounds that modulate AGC protein kinases and are suitable for the preparation of pharmaceutical compositions for oral, parenteral, topical, rectal, nasal, buccal, vaginal administration or via an implanted reservoir or by inhalation spray. A further object is to provide compounds that modulate AGC protein kinases having a PIF binding pocket in the N-terminal lobe of the catalytic domain, such as PKCzeta and SGK. A further object is to provide compounds which activate PDK1 and inhibit PDK1 and/or PKB. A further object is to provide pharmaceutical compositions suitable for treating diseases associated with protein kinases, in particular AGC kinases, PDK1 signalling and PKB signalling, e.g. cancer or diabetes.